Figure 1
A, Growth curves of VMM18 melanoma cells in 5% and 0.5% serum-containing media. VMM18 melanoma cells were cultured in media containing either 5% or 0.5% serum and were assayed in triplicate at times 0, 4, 8, 16, 24, 48, and 72 hours using Cell Titer 96 Aqueous (Promega; Madison, WI) according to the directions supplied by the manufacturer. The absorbance at 490 nm (OD 490) measures the quantity of formazan product from the MTS-based assay and is directly proportional to the number of live cells present. The R2 values for the linear regression lines determined using Microsoft Excel are listed above each line. The solid dark line with the squares represents the data collected from VMM18 melanoma cells grown in media with 5% serum. The thin line with the circles represents the data from VMM18 melanoma cells grown in media containing 0.5% serum. B, Western blot analysis of 4EBP1 from melanoma cell lines grown in 5.0% FBS and 0.5% FBS. Phosphorylation of 4EBP1 was assayed by its reduced migration in SDS-PAGE and the proteins were detected by immunoblotting. VMM5A, VMM18, and VMM39 cells were grown in media as indicated. C, Western blot analysis of ERK from melanoma cell lines grown in 5.0% FBS and 0.5% FBS. The dual phosphorylation of ERK was analyzed by phosophosite-specific immunoblotting in VMM5A, VMM18, and VMM39 melanoma cells cultured as described (upper panel). The total amount of ERK protein was determined by immunoblotting with a separate antibody (lower panel). The relative phosphorylation of ERK was quantitated by densitometry analysis using Image Quant 5.2 software and the values are given below the top panel.